The present invention relates to a process for making nucleosides, nucleoside analogs and derivatives. The invention has particular application to the enzymatic synthesis of nucleosides, nucleoside analogs and derivatives.
Nucleoside analogs have been used extensively as antibiotic substances and as biological probes. See Suhadolnik, Nucleoside Antibiotics, J. Wiley, New York, 1970; Suhadolnik, Nucleosides as Biological Probes, J. Wiley, New York, 1979; Nucleoside Analogues; Chemistry, Biology and Medicinal Applications, Walker, et al., Eds., NATO Advanced Study Institutes Series, Plenum, New York, 1979, Vol. 26. Recent interest in this class of compounds has been stimulated by the efficacy of certain nucleosides as anti-parasitic and antiviral agents. See Hupe, Ann. Reports in Medicinal Chemistry, Bailey, Ed., Academic Press, New York, 1986, Vol. 21., Ch. 23; Mansuri, et al., Ann. Reports in Medicinal Chemistry, Bailey, Ed., Academic Press, New York, 1987, Vol. 22, Ch. 15; Mansuri, et al., Ann Reports in Medicinal Chemistry, Allen, Ed., Academic Press, New York, 1988, Vol. 23, Ch. 17. Zidovudine (3'-azido-3'-deoxythymidine, AZT) and the various 2',3'-dideoxynucleosides have received special attention because of their virucidal activity in the treatment of AIDS patients See Ezzell, Nature, 1987, Vol. 326, p. 430; DeClerq, Trends in Pharmacol. Sci., 1987, Vol. 8, pp. 339-45. The broad-spectrum antiviral activity of 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (hereinafter referred to as RTCA) has recently been shown to extend to the treatment of plant as well as animal viruses. See Lerch, Antiviral Res., 1987, Vol. 7, pp. 257-70.
Traditionally, nucleosides have been prepared by various chemical methods, including those described in Nucleoside Analogues; Chemistry, Biology and Medicinal Applications, Walker, et al., Eds., NATO Advanced Study Institutes Series, Plenum, New York, 1979, Vol. 26. Recently, however, a number of papers have appeared reporting the enzymatic preparations of both natural and unnatural nucleosides. See, for example, Krenitsky, et al., J. Med. Chem., 1986, Vol. 29, pp. 138-143; Utagawa, et al., Agric. Biol. Chem., 1986, Vol. 50(1), pp. 121-126; Krenitsky, et al., Carbohydrate Research, 1981, Vol. 97, pp. 139-146, Krenitsky, et al., Biochemistry, 1981, Vol. 20, pp. 3615-3621. Many of those papers report transglycosylation reactions wherein the original heterocyclic base is exchanged for a new aglycon moiety. Those works employed two basic strategies.
The first strategy involved the enzymatic preparation of ribose-1-phosphate ("R-1-P") from a nucleoside, followed by the isolation of the R-1-P. The isolated R-1-P was then used as the glycosyl donor in an enzymatic coupling reaction with added heterocycles. An overall purine to purine analog exchange could be accomplished by this means.
The second strategy used a pyrimidine nucleoside as the glycosyl donor and a purine or purine analog as the glycosyl acceptor. This was conducted as a one-pot reaction without the isolation of R-1-P but required that both a pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase be present in the reaction media.
Of interest here is the report by Utagawa, et al., Agric. Biol. Chem., 1986, Vol. 50(1), pp. 121-126, that the purine analog 1,2,4-triazole-3-carboxamide ("TCA", the aglycon component of RTCA) could not be glycosylated to any measurable extent in a one-pot reaction using inosine as the glycosyl donor and purine nucleoside phosphorylase ("PNPase") as the catalyst. Those workers cited the low affinity of TCA (K.sub.m =167 mM) compared to the affinity of hypoxanthine (the phosphorolysis product of inosine, K.sub.m =5.6 mM) for PNPase. Hypoxanthine perhaps acts as a competitive inhibitor of RTCA synthesis.
The disadvantages of the previous enzymatic strategies are that they require either the isolation of R-1-P or the presence of both pyrimidine and purine nucleoside phosphorylases. There is a need for a new synthetic method that overcomes those restrictions. The process of the present invention provides such a method.